Research project Cell Biology of nutrient regulated gene expression
Proteins with multiple membrane-segments (MS) initially insert into the endoplasmic reticulum (ER) of eukaryotic cells. Little is known how such proteins attain native functional structures. We study the ER membrane chaperone Shr3 that specifically facilitates the folding of all 18 members of the yeast amino acid permease family of proteins with 12 MS.
Shr3 is an integral component of the ER that is specifically required for functional expression of an entire family of nutrient transport proteins, i.e., amino acid permeases (AAP). Although the precise function of Shr3 and its high degree of selectivity for AAP remains mysterious, it is known that in the absence of Shr3 (shr3Δ), AAP correctly insert via the Sec61 translocon and each MS segment partitions into the ER membrane but they misfold, forming high molecular weight aggregates that are retained in the ER. The aggregates are eventually degraded in an ERAD-dependent manner. We know that Shr3 productively engages with the first 5 MS (I-V) of split Gap1, and facilitates the subsequent assembly of an independently expressed C-terminal 7 MS (VI-XII) split-Gap1. Based on these findings, it can be inferred that Shr3 interacts with nascent N-terminal MS of AAP, an interaction that is essential for folding of the holoprotein. Together with the ability of Shr3 to interact with COPII components via its C-terminal cytoplasmic tail the biological role of Shr3 is apparently a nexus between AAP folding and packaging into COPII-coated vesicles.
In this project we are attempting to fill gaps in understanding regarding the requirement for Shr3 during the co-translational folding of AAP. We have focused on the membrane domain of Shr3 and employed a comprehensive scanning mutagenesis approach to define amino acid residues involved in recognizing AAP substrates. Further, we have exploited a modified split-ubiquitin approach to directly probe and characterize interactions with seven different AAP substrates in vivo. The data support that Shr3 acts as a membrane-localized chaperone (MLC) specifically required to help nascent chains of partially translated AAP attain and maintain a structure required to follow a productive folding pathway as translation proceeds to completion. Shr3 does not engage in productive interactions with similarly complex membrane proteins that belong to the sugar transporter protein family.
Ongoing work is directed at obtaining a precise map of Shr3-AAP interactions that likely guide the folding AAP as the nascent chains emerge from the Sec61 translocon. Here we are applying the unnatural amino acid mutagenesis technique developed by Schultz and coworkers to site-specifically insert photoactive amino acids into the Shr3 sequence. Ultimately, we should obtain living cells expressing homogenous populations of Shr3 with precisely positioned cross-linkable amino acids. Analysis of the crosslinked products in these cells should provide detailed information regarding the mechanisms of membrane insertion and the timing of Shr3 interactions.